Completing the heterotrimer:: Isolation and characterization of an Arabidopsis thaliana G protein γ-subunit cDNA

Heterotrimeric G proteins consist of three subunits (alpha, beta ,and gamma). alpha- and beta- subunits have been previously cloned in plants, but the gamma -subunit has remained elusive. To isolate the gamma -subunit of a plant heterotrimeric G protein an Arabidopsis thaliana yeast two-hybrid library was screened by using a tobacco G-beta -subunit as the bait protein. One positive clone (AGG1) was isolated several times; it displays significant homology to the conserved domains of mammalian gamma -subunits. The predicted AGG1 protein sequence contains all of the typical characteristics of mammalian gamma -subunits such as small size (98 amino acids, 10.8 kDa), presence of a C-terminal CAAX box to direct isoprenyl modification, and an N-terminal alpha -helix region capable of forming a coiled-coil interaction with the beta -subunit. Northern and Southern analyses showed that AGG1 is a single-copy gene in Arabidopsis with a similar expression pattern to the Arabidopsis beta -subunit, AGB1 [Weiss, C. A., Garnaat, C. W., Mukai, K., Hu, Y. & Ma, H. (1994) Proc Natl. Acad. Sci. LISA 91, 9554-9558]. By using the yeast two-hybrid system, we show that AGG1 strongly interacts with tobacco and Arabidopsis beta -subunits. The in vivo results have been confirmed by using in vitro methods to prove the interaction between AGG1 and the Arabidopsis beta -subunit As previously observed in mammalian systems, both the coiled-coil domain and the WD repeat regions of the beta -subunit are essential for AGG1 interaction. Also in agreement with previous observations, the removal of the N-terminal alpha -helix of the AGG1 greatly reduces but does not completely block the interaction.